Taxol, a complex diterpene from Taxus brevifolia (Pacific Yew), has shown excellent potential as an anti-tumor chemotherapeutic agent, especially against ovarian cancer. It acts by binding to microtubules imparting hyperstability to them. Although some studies on the interaction of taxol and some of its derivatives with tubulin and microtubules have been done, nothing is known about its binding site or domain in tubulin. Such information is useful for the further development of active taxol derivatives. The overall aim of this proposal is to synthesize photoaffinity analogues of taxol and to use them to identify peptides in the taxol binding site. The work is divided into three parts. We plan to synthesize phenylazido, nitrophenylazido, and trifluoroethyl benzoyl diazirine derivatives of taxol. These functionalities will be placed at different positions of the baccatin III and phenylisoserine side chain moieties. Not all proposed compounds will be synthesized if we find useful ones early in the work. The taxol derivatives will be compared to taxol in regard to their activities in the microtubule assembly reaction in vitro and on B16 melanoma cells in culture. The binding of the active compounds to microtubules and tubulin will also be measured. The analogues with good activities and binding will be used to label tubulin. This will require the synthesis of radiolabeled analogues. Various digestion and separation procedures together with peptide sequencing and mass spectrometry will be used to identify peptides containing covalently bound analogue.